Planta (1983)159:404-410
P l a n t a 9 Springer-Verlag 1983
Regulation and characterization of two inducible amino-acid transport systems in Chlorella vulgaris Norbert Sauer 1, Ewald Komor 2 and Widmar Tanner a 1 Institut fiir Botanik der Universit/it, Universit/itsstrasse 31, D-8400 Regensburg, and 2 Institut ffir Botanik der Universit/it, Universitfitsstrasse 30, D-8580 Bayreuth, Federal Republic of Germany
Abstract. Glucose or non-metabolizable glucose analogues induce two amino-acid transport systems in Chlorella vulgaris: an arginine system (arginine and lysine) and a proline system (proline, glycine, alanine and serine). The same amino-acid transport systems are induced in the absence of glucose, when the cells are depleted of their nitrogen source as judged by a comparison of K m values and the lack of additive induction by the two treatments. Changes in the concentration of neither internal free amino acids nor of soluble carbohydrate pools correlate perfectly with the induction of amino-acid transport. Also exogenous cAMP had no effect on the induction of transport. Both aminoacid transport systems are able to accumulate free amino acids more than 1000-fold. The accumulation plateau is not due to a steady state of influx and efflux, but rather arises by a shut-off of influx. No significant efflux is observed. The biological importance of this frequently observed behaviour in amino-acid transport is discussed. Key words: Amino acid (transport and accumulation) - Chlorella - Nitrogen starvation - Transport induction (amino acid) - Transport regulation (amino acid).
Introduction
A large variety of cells from lower and higher plants have been shown to possess transport systems which catalyze transmembrane translocation of sugars or amino acids (Komor 1982; Raven 1980; Colombo et al. 1978; Felle 1981; Guy et al. 1981). Only a few such systems have been reported, however, which change their activity dramatically as a result of an induction-like effect of the type
which has often been reported for bacteria (Cohen and Monod 1957; Oxender 1972). Hexose transport in chlorococcal algae, especially in Chlorella vulgaris, is one such example (Tanner 1969, Haag and Tanner 1974). Surprisingly, hexose and hexose analogues in Chlorella vulgaris not only induce ~ a hexose transport system, but also two amino-acid transport systems (Cho et al. 1981). One of these is specific for alanine, glycine, serine, and proline and will be called the proline system. The other one is specific for the basic amino acids, arginine and lysine, and will be called the arginine system. Both these systems are able to transport the corresponding amino acids against a high gradient of free internal amino acids (Cho et al. 1981) and the proline system, at least, has been shown to be a H+-symport system (Cho and Komor, unpublished data). In this paper results are presented to show that the same two amino-acid transport systems can also be induced by nitrogen shortage. In addition, the capability of the cells to accumulate amino acids under changing outside concentrations has been studied, and finally, the question was investigated whether the accumulation plateaus are caused by compensation of influx by efflux (Komor and Tanner 1971) or by "shut-off" (Crabeel and Grenson 1970). Material and methods Plant material. The strain of Chlorella vulgaris and the growth conditions were the same as described previously (Tanner and Kandler 1967). The vacuolation of this strain of C. vulgaris amounts to less than 10% of the total volurne (Komor, unpublished). For experiments with N-starved cells the algae were 1 The term induction is used in this paper for the phenomenon of a greatly increased transport activity without implications on possible mechanisms
405
N. Sauer et al. : Amino-acid transport in algae harvested, washed once with distilled water and transferred to a nitrogen-free medium. All the other conditions remained unchanged. Uptake experiments with N-starved cells were carried out after a 24-h period in this medium.
Table 1. Effect of glucose preincubation and of nitrogen starvation on the rate of amino-acid uptake in Chlorella Amino acid
Rate of uptake (gmoles h 1 m l - 1 packed cells)
Chemicals. D-[U-14C]glucose (specific activity 1.206"1013 Bqtool-l), L_[U 14C]arginine (specific activity 1.263.1013 Bqmol-1), t-[U-i4C]proline (specific activity 1.093.1013Bq tool -1) and a L-[U-14C]amino acid kit (specific activity 3.77.101I Bq.mol 1 each) were purchased from New England Nuclear (Boston, Mass., USA).
Glucose treatment of the cells. For glucose treatment, the cells from normal cultures or from N-starved cultures were harvested, washed with distilled water and resuspended to a cell density of 50 gl packed cells m l - 1 in 25 m M sodium-phosphate buffer, pH 6.0. These cells were shaken for 3 h at 27~ in the presence of 13 m M D-glucose. Cells not treated with glucose were shaken for the same time without addition of sugar.
Uptake experiments. Uptake experiments were carried out in 25 mM sodium-phosphate buffer, pH 6.0, at cell densities between 10 and 20 gl packed cells ml-1. The cell suspension was shaken in a water bath at 27 ~ C in the dark. The experiment was started by the addition of radioactive substrate to the cells. The final substrate concentration, if not indicated otherwise, was 1 mM; its specific activity was 6.41.10 9 Bq-mol-1. Every 30 s, for 3 min a 100-~tl sample of the suspension was withdrawn, filtered on nitrocellulose filters (Schleicher & Schfill, Dassel, F R G ; pore size 0.8 gin) and washed with 10 ml of icecold sodium-phosphate buffer (25 mM, pH 6.0). Cells and filter were added to 5 ml scintillation cocktail (5 g 2,5-diphenyloxazole (PPO) and 100 g naphthalene per I of dioxane) and counted in a Beckman LS-7000 Counter (Munich, FRG).
Determination of gross influx and efflux (=unidirectional fluxes). For the determination of gross influx and gross efflux at various times along a net uptake curve, two parallel samples (called A and B) of induced algae were incubated with 1 mM amino acids. 14C-Labeled amino acid was added to sample A. At the selected time, portions of both sample A and B were centrifuged at 3000 g (Hettich Rotina S, Tuttlingen, FRG) for 5 min and the pellet of one sample was resuspended with the supernatant of the other. In this way the conditions for measuring net and gross uptake are essentially identical. Uptake of radioactivity in sample B ( = gross uptake) was measured immediately as described above; efflux of radioactivity in sample A ( = gross efflux) was followed for at least 30 min (six measurements) by spinning down 200-gl samples for 20 s at 10,000 g (Eppendorf 5412, Hamburg, FRG) and pipetting 100 ~tl of the supernatant into 5 ml scintillation cocktail.
Preparation of extracts. Packed cells (200 gl) were boiled with 5 ml distilled water for 5 rain and centrifuged for 20 rain at 50,000 g. For sugar analysis, the supernatant was evaporated to dryness and after resolution in a small volume of distilled water, desalted via Dowex-columns (Serva, Heidelberg, F R G ; Dowex 50 WX 8 (100-200) and Dowex 1 x 2 acetate form (100-200), bed volume I ml of each).
Sugar analyses. Sugars were analyzed with a gas chromatograph (Hewlett-Packard 5830 A) on a 3 % SP-2340 column after acetylation by the method of Albersheim (1967). Sucrose was determined as hexose, which is liberated by incubation in either 0.1 N HzSO 4 at 10W C for 10 rain or with invertase (10 mg m1-1 in 10 m M acetate-buffer, pH 4.5) at room temperature for 45 min.
Cells not pretreated
Cells N-starved pretreated cells with glucose
Group I 5-Arginine L-Lysine
0.53 1.22
86 29
84 32
L-Proline L-Serine L-Alanine Glycine
0.36 0.28 0.52 0.75
66 90 78 79
32 37 25 31
Group II L-Asparagine L-Aspartic acid c-Glutamic acid L-Cysteine L-Phenylalanine L-Tyrosine c-Tryptophan L-Isoleucine L-Valine L-Leucine
0.42 0.41 0.39 0.55 0.26 0.16 0.01 0.16 0.17 0.31
0.42 0.42 0.39 0.50 0.16 0.16 0.01 0.16 0.20 1.02
0.45 0.57 2.25 n.d? 0.41 0.60 n.d." 0.43 0.80 0.68
Group III L-Histidine L-Glutamine L-Methionine L-Threonine
0.05 0.68 0.07 0.25
3.2 5.1 0.6 2.1
4.2 0.76 5.8 0.7
" Not determined
Amino acid analyses. The amino-acid analyses of the hot-water extract were performed with an amino-acid analyzer (Kontron, Zfirich, Switzerland) on the cation exchange resin DC - 6 A (Durrum, Palo Alto, USA). The amino acids were detected with ninhydrin at 570 nm and 440 nm.
Results
Glucose and to some extent also non-metabolizable glucose analogues induce two amino-acid uptake systems in Chlorella vulgaris (Cho et al. 1981). When Chlorella cells were kept for 24 h in a nitrogen-free medium, they became yellowish green. These cells possessed an increased capability to take up some amino acids. Thus per ml packed cells the uptake of L-serine, L-proline, glycine and L-alanine increased 41- to 132-fold (Table 1). The rate of uptake of L-arginine and L-lysine behaved similarly. When 18 amino acids of the 20 occurring in proteins were tested, it became quite obvious that their rates of uptake were affected by nitrogen starvation in a way resembling glucose pretreatment (Table 1). The amino acids of group II (Table 1) were not transported at an increased rate
406
N. Sauer et al. : Amino-acid transport in algae
Table 2. Effect of glucose or glucose plus nitrate on the rate of arginine uptake and on the internal concentration of soluble carbohydrates and amino acids in Chlorella. In the glucose + nitrate sample, 10 m M N O 3 were present; other conditions see Material and methods Cellular concentration (raM) of
Time after glucose addition (h)
0
1
Time after glucose + N O addition (h)
2
1
2
Sucrose Glucose
4.22 1.11
4.80 1.73
9.92 1.20
4.11 1.22
8.95 2.60
Arginine Asparagine Glutamic acid + glutamine Total amino acids
2.88 6.25 7.34
2.61 5.01 10.42
2.32 2.77 7.45
2.64 5.58 11.70
2.82 6.51 12.75
35.57
35.38
30.92
36.96
43.49
6.5
60.7
77.9
94.4
Transport of arginine (pmol h 1 m l - i packed cells)
121.0
neither after nitrogen starvation nor after glucose pretreatment. The uptake rates of the amino acids of group III, except for glutamine, and threonine showed a large increase (e.g. methionine 80-fold), but the absolute rates were quite small compared with those of group I. As with glucose-treated cells, it is not clear, therefore, whether specific transport systems for methionine and histidine are "induced" by nitrogen starvation, or whether these amino acids are taken up to some extent by the very active proline and arginine system. The question arises as to whether a common factor is responsible for the increased activity of the amino-acid transport systems under these two different conditions. Nitrogen-starved cells would be expected to possess only small pools of free amino acids. High amounts of soluble carbohydrates would be expected for glucose-fed cells, since almost 50% of the glucose taken up is converted to sucrose (Tanner et al. 1966). In the case of induction by the non-metabolizable analogue 6-deoxy-glucose (Cho et al. 1981), this sugar analogue at least is accumulated to high levels within the cells (Komor et al. 1973). Whether the cellular concentrations of amino acids decrease during glucose or 6-deoxyglucose treatment or the concentrations of soluble carbohydrates increase during nitrogen starvation is not known. To find possible correlations of the increased activity of the aminoacid transport systems with internal concentrations of free sugars and amino acids, changes in concen-
Table 3. Effect of N-starvation on the rate of arginine uptake
and on internal concentration of soluble carbohydrates and amino acids in Chlorella
Exp. I
Cellular concentration (mM)
Time in N-free medium (h) 0
3.5
5
12
Sucrose Arginine Asparagine Glutamic acid + glutamine Total amino acids
2.90 2.35 5.76 3.98
8.66 0.98 1.51 2.60
9.13 0.53 0.86 0.86
-
28.48
20.34
13.46
-
2.5
15.1
21.4
-
1.09 0.21
-
-
13.58 1.18
3.0
-
-
45.5
Transport of arginine (gmol h - i m l - i packed cells) Exp. II Sucrose Glucose Transport of arginine (pmol h - 1 ml 1 packed cells)
tration of these compounds under the various conditions were followed. In Table 2, the cellular concentrations of glucose, sucrose, and amino acids at different times after addition of glucose or glucose plus nitrate are listed. Whereas the arginine transport rate increased 10- to 15-fold within 1 h of glucose treatment, neither a significant increase in sugars nor a corresponding decrease in amino acids has been observed. Also, none of the other amino acids which are not listed in Table 2 changed its concentration during the first hour. Two hours after glucose addition, the concentration of sucrose had doubled and asparagine had decreased to about half. When induction with glucose, however, is carried out in the presence of nitrate, neither the decrease in asparagine nor in total amino acids can be observed, whereas the change in sucrose concentration remains unaffected. But in spite of it, sucrose cannot be regarded as a possible inducer, because its concentration clearly increases subsequent to the induction of the arginine transporter. The same holds for glucose, galactose and mannose (data partly not shown). Under nitrogen starvation (Table 3), on the other hand, the increase in the rate of arginine uptake correlates well with the decrease in concentration of some amino acids, like asparagine, glutamine plus glutamic acid and arginine. This is also true for the increase in sucrose content. Amino acids and sucrose change in concentration before
N. Sauer et al. : Amino-acid transport in algae ,F:
'-2 u
"7 E
% -,D 10
'2O
IE
407 Table 4. Transport of arginine in Chlorella. Comparison of Km and Vmax values. Uptake experiments were performed as described under Material and methods. Cell densities varied from 10 gl packed cells m1-1 at 1 mM L-arginine (specific activity: 6.41.109 Bq-mo1-1) to 0.01 gl packed cells/ml at 0.25 pM Larginine (specific activity: 1.282"1013Bq-mol-1). The Vmax values are average values of 7 experiments Ceils N-starved pretreated cells with glucose
o u
"G o~
5
Km(gM) Vmax (gmol ml- 1 packed cells h - 1)
1.6 104 • 31
1.6 73 _+19
u c o u
'~
0
0
I
2
3
6
g '-
5
Time [ h I
Fig. 1. Comparison of changes in cellular concentrations of asparagine ( o - - o ) , and sucrose ( n - - n ) with the increasing rate for arginine transport (e e). At zero time the Chlorella cells were transferred to N-free medium in the light plus CO 2 ; p.c. = packed cells
a significant induction of arginine uptake can be observed. In the first h o u r after the removal of the N-source, the concentration of asparagine drops to 50% o f the previous value while sucrose doubles (Fig. 1). Thus, it could be concluded that under nitrogen starvation the internal pools of amino acids or sucrose, or some kind of a C / N ratio might control the induction o f the two aminoacid transport systems. Since induction by glucose does not result in the same correlative pattern, the question arose, whether the increased rate o f amino-acid uptake after N-starvation and glucose pretreatment is due to identical systems.
Comparison of arginine transport induced in two different ways. First, the specificity of the differently induced arginine transporters was examined. As already mentioned above (Table 1), with the exception of small differences, the same amino acids are transported at a high rate after both m e t h o d s of induction. Second, in view o f the fact that an i m p o r t a n t aspect concerning the identity of the two transport proteins would be identical K m values, the apparent K m values and the Vm,~ values for arginine uptake o f glucose-treated and N-starved cells are compared (Table 4). The K m values o f 1.6 g M are identical under both conditions, the Vm, ~ values are generally 20-30% lower in N-starved cells. A lowered rate of protein synthesis in these cells might be responsible for this difference.
Table 5. Effect of glucose treatment on transport rates (pmol ml-1 packed cells h-1) of nitrogen-starved cells of Chlorella. Where indicated 10 mM NO~- was present; other conditions see Material and methods Transported compound
N-starved cells (control)
Cells treated with glucose
Cells treated with glucose+ NO3-
Glucose Arginine Proline
0.7 57.0 59.5
6.5 45.6 47.6
47.6 62.7 41.7
Finally, it was investigated whether the two transporters can be induced additively. In Nstarved cells the hexose uptake system of Chlorella is not active. Addition of glucose to such cells also does n o t increase glucose uptake significantly. This indicates that shortage o f internal amino acids limits the synthesis of the hexose transport protein (Fenzl et al. /977). U n d e r these conditions glucose also would not be expected to have any inducing effect on the amino-acid uptake systems. This is indeed the case (Table 5). W h e n these cells are treated, however, with glucose plus nitrate, the rate of glucose uptake increases almost 70-fold (alt h o u g h the rate obtained is only 25-30% of that observed in normal cells after induction). This shows that under these conditions the cells are capable of de-novo protein synthesis started by an inducer such as glucose. U n d e r the same conditions, however, there is no increase in the activity of the arginine transport system (Table 5). Thus, the two treatments, glucose induction and nitrogen starvation are not additive, which indeed supports the conclusion that one and the same transport system is being turned o n by the two treatments.
Comparison of the proline transport system induced in two different ways. The proline uptake system was characterized in the same way as arginine uptake. In Table 6, the apparent K m and the Vm~x
408
N. Sauer et al. : Amino-acid transport in algae
Table 6. Transport of proline in Chlorella. Comparison of K m and Vm~X values. The data were obtained as described in Table 4. The Vm~x values are average values of five experiments
u
"T =o
100
Cells N-starved pretreated cells with glucose K m (p.M) V,.ax (pmol ml 1 packed cells h - 1)
39 94_+22
50
35 47 _+12
I
values for glucose-pretreated and N-starved cells are compared. There was no significant difference between cells treated in either way. The K m value of around 40 gM is, however, more than a factor of 20 higher than that for arginine. As with the arginine system, the Vmax of proline uptake by glucose-induced cells is generally somewhat higher than that of N-starved cells. The fact that the proline system cannot be additively induced (Table 5), and that it shows the same specificity after both methods of induction (Table 1), leads to the same conclusion as with the arginine system: in both cases it is likely that the same transport protein becomes induced.
How does the maximal inside concentration during proline and arginine uptake arise? Uptake of arginine proceeds beyond the concentration equilibrium (Cho et al. 1981). This is demonstrated in greater detail in Table 7. Accumulation ratios of 600- and up to 1200-fold are observed at an initial outside concentration of I m M (see also Cho et al. 1981). The accumulation ratio decreases with increasing outside concentration, a phenomenon generally observed in transport physiology (Komor etal. 1973; Kotyk 1973; Eddy 1982). The maximal accumulation ratios observed are far beyond the electrochemical equilibrium potential of about 130 mV inside negative (Komor and Tanner 1976) and thus not in agreement with a mechanism of arginine moving as a cation in a uniport manner; they rather indicate an Arg +/H + symport.
[
I
I
I
I
60
120 Time [ min]
Flux Rates
[ y m o l h -ImL-Ip.c.]
Net ]nftux
Gross Influx
rain
180
180
rain
57
61
P l a n t a 9 Springer-Verlag 1983
Regulation and characterization of two inducible amino-acid transport systems in Chlorella vulgaris Norbert Sauer 1, Ewald Komor 2 and Widmar Tanner a 1 Institut fiir Botanik der Universit/it, Universit/itsstrasse 31, D-8400 Regensburg, and 2 Institut ffir Botanik der Universit/it, Universitfitsstrasse 30, D-8580 Bayreuth, Federal Republic of Germany
Abstract. Glucose or non-metabolizable glucose analogues induce two amino-acid transport systems in Chlorella vulgaris: an arginine system (arginine and lysine) and a proline system (proline, glycine, alanine and serine). The same amino-acid transport systems are induced in the absence of glucose, when the cells are depleted of their nitrogen source as judged by a comparison of K m values and the lack of additive induction by the two treatments. Changes in the concentration of neither internal free amino acids nor of soluble carbohydrate pools correlate perfectly with the induction of amino-acid transport. Also exogenous cAMP had no effect on the induction of transport. Both aminoacid transport systems are able to accumulate free amino acids more than 1000-fold. The accumulation plateau is not due to a steady state of influx and efflux, but rather arises by a shut-off of influx. No significant efflux is observed. The biological importance of this frequently observed behaviour in amino-acid transport is discussed. Key words: Amino acid (transport and accumulation) - Chlorella - Nitrogen starvation - Transport induction (amino acid) - Transport regulation (amino acid).
Introduction
A large variety of cells from lower and higher plants have been shown to possess transport systems which catalyze transmembrane translocation of sugars or amino acids (Komor 1982; Raven 1980; Colombo et al. 1978; Felle 1981; Guy et al. 1981). Only a few such systems have been reported, however, which change their activity dramatically as a result of an induction-like effect of the type
which has often been reported for bacteria (Cohen and Monod 1957; Oxender 1972). Hexose transport in chlorococcal algae, especially in Chlorella vulgaris, is one such example (Tanner 1969, Haag and Tanner 1974). Surprisingly, hexose and hexose analogues in Chlorella vulgaris not only induce ~ a hexose transport system, but also two amino-acid transport systems (Cho et al. 1981). One of these is specific for alanine, glycine, serine, and proline and will be called the proline system. The other one is specific for the basic amino acids, arginine and lysine, and will be called the arginine system. Both these systems are able to transport the corresponding amino acids against a high gradient of free internal amino acids (Cho et al. 1981) and the proline system, at least, has been shown to be a H+-symport system (Cho and Komor, unpublished data). In this paper results are presented to show that the same two amino-acid transport systems can also be induced by nitrogen shortage. In addition, the capability of the cells to accumulate amino acids under changing outside concentrations has been studied, and finally, the question was investigated whether the accumulation plateaus are caused by compensation of influx by efflux (Komor and Tanner 1971) or by "shut-off" (Crabeel and Grenson 1970). Material and methods Plant material. The strain of Chlorella vulgaris and the growth conditions were the same as described previously (Tanner and Kandler 1967). The vacuolation of this strain of C. vulgaris amounts to less than 10% of the total volurne (Komor, unpublished). For experiments with N-starved cells the algae were 1 The term induction is used in this paper for the phenomenon of a greatly increased transport activity without implications on possible mechanisms
405
N. Sauer et al. : Amino-acid transport in algae harvested, washed once with distilled water and transferred to a nitrogen-free medium. All the other conditions remained unchanged. Uptake experiments with N-starved cells were carried out after a 24-h period in this medium.
Table 1. Effect of glucose preincubation and of nitrogen starvation on the rate of amino-acid uptake in Chlorella Amino acid
Rate of uptake (gmoles h 1 m l - 1 packed cells)
Chemicals. D-[U-14C]glucose (specific activity 1.206"1013 Bqtool-l), L_[U 14C]arginine (specific activity 1.263.1013 Bqmol-1), t-[U-i4C]proline (specific activity 1.093.1013Bq tool -1) and a L-[U-14C]amino acid kit (specific activity 3.77.101I Bq.mol 1 each) were purchased from New England Nuclear (Boston, Mass., USA).
Glucose treatment of the cells. For glucose treatment, the cells from normal cultures or from N-starved cultures were harvested, washed with distilled water and resuspended to a cell density of 50 gl packed cells m l - 1 in 25 m M sodium-phosphate buffer, pH 6.0. These cells were shaken for 3 h at 27~ in the presence of 13 m M D-glucose. Cells not treated with glucose were shaken for the same time without addition of sugar.
Uptake experiments. Uptake experiments were carried out in 25 mM sodium-phosphate buffer, pH 6.0, at cell densities between 10 and 20 gl packed cells ml-1. The cell suspension was shaken in a water bath at 27 ~ C in the dark. The experiment was started by the addition of radioactive substrate to the cells. The final substrate concentration, if not indicated otherwise, was 1 mM; its specific activity was 6.41.10 9 Bq-mol-1. Every 30 s, for 3 min a 100-~tl sample of the suspension was withdrawn, filtered on nitrocellulose filters (Schleicher & Schfill, Dassel, F R G ; pore size 0.8 gin) and washed with 10 ml of icecold sodium-phosphate buffer (25 mM, pH 6.0). Cells and filter were added to 5 ml scintillation cocktail (5 g 2,5-diphenyloxazole (PPO) and 100 g naphthalene per I of dioxane) and counted in a Beckman LS-7000 Counter (Munich, FRG).
Determination of gross influx and efflux (=unidirectional fluxes). For the determination of gross influx and gross efflux at various times along a net uptake curve, two parallel samples (called A and B) of induced algae were incubated with 1 mM amino acids. 14C-Labeled amino acid was added to sample A. At the selected time, portions of both sample A and B were centrifuged at 3000 g (Hettich Rotina S, Tuttlingen, FRG) for 5 min and the pellet of one sample was resuspended with the supernatant of the other. In this way the conditions for measuring net and gross uptake are essentially identical. Uptake of radioactivity in sample B ( = gross uptake) was measured immediately as described above; efflux of radioactivity in sample A ( = gross efflux) was followed for at least 30 min (six measurements) by spinning down 200-gl samples for 20 s at 10,000 g (Eppendorf 5412, Hamburg, FRG) and pipetting 100 ~tl of the supernatant into 5 ml scintillation cocktail.
Preparation of extracts. Packed cells (200 gl) were boiled with 5 ml distilled water for 5 rain and centrifuged for 20 rain at 50,000 g. For sugar analysis, the supernatant was evaporated to dryness and after resolution in a small volume of distilled water, desalted via Dowex-columns (Serva, Heidelberg, F R G ; Dowex 50 WX 8 (100-200) and Dowex 1 x 2 acetate form (100-200), bed volume I ml of each).
Sugar analyses. Sugars were analyzed with a gas chromatograph (Hewlett-Packard 5830 A) on a 3 % SP-2340 column after acetylation by the method of Albersheim (1967). Sucrose was determined as hexose, which is liberated by incubation in either 0.1 N HzSO 4 at 10W C for 10 rain or with invertase (10 mg m1-1 in 10 m M acetate-buffer, pH 4.5) at room temperature for 45 min.
Cells not pretreated
Cells N-starved pretreated cells with glucose
Group I 5-Arginine L-Lysine
0.53 1.22
86 29
84 32
L-Proline L-Serine L-Alanine Glycine
0.36 0.28 0.52 0.75
66 90 78 79
32 37 25 31
Group II L-Asparagine L-Aspartic acid c-Glutamic acid L-Cysteine L-Phenylalanine L-Tyrosine c-Tryptophan L-Isoleucine L-Valine L-Leucine
0.42 0.41 0.39 0.55 0.26 0.16 0.01 0.16 0.17 0.31
0.42 0.42 0.39 0.50 0.16 0.16 0.01 0.16 0.20 1.02
0.45 0.57 2.25 n.d? 0.41 0.60 n.d." 0.43 0.80 0.68
Group III L-Histidine L-Glutamine L-Methionine L-Threonine
0.05 0.68 0.07 0.25
3.2 5.1 0.6 2.1
4.2 0.76 5.8 0.7
" Not determined
Amino acid analyses. The amino-acid analyses of the hot-water extract were performed with an amino-acid analyzer (Kontron, Zfirich, Switzerland) on the cation exchange resin DC - 6 A (Durrum, Palo Alto, USA). The amino acids were detected with ninhydrin at 570 nm and 440 nm.
Results
Glucose and to some extent also non-metabolizable glucose analogues induce two amino-acid uptake systems in Chlorella vulgaris (Cho et al. 1981). When Chlorella cells were kept for 24 h in a nitrogen-free medium, they became yellowish green. These cells possessed an increased capability to take up some amino acids. Thus per ml packed cells the uptake of L-serine, L-proline, glycine and L-alanine increased 41- to 132-fold (Table 1). The rate of uptake of L-arginine and L-lysine behaved similarly. When 18 amino acids of the 20 occurring in proteins were tested, it became quite obvious that their rates of uptake were affected by nitrogen starvation in a way resembling glucose pretreatment (Table 1). The amino acids of group II (Table 1) were not transported at an increased rate
406
N. Sauer et al. : Amino-acid transport in algae
Table 2. Effect of glucose or glucose plus nitrate on the rate of arginine uptake and on the internal concentration of soluble carbohydrates and amino acids in Chlorella. In the glucose + nitrate sample, 10 m M N O 3 were present; other conditions see Material and methods Cellular concentration (raM) of
Time after glucose addition (h)
0
1
Time after glucose + N O addition (h)
2
1
2
Sucrose Glucose
4.22 1.11
4.80 1.73
9.92 1.20
4.11 1.22
8.95 2.60
Arginine Asparagine Glutamic acid + glutamine Total amino acids
2.88 6.25 7.34
2.61 5.01 10.42
2.32 2.77 7.45
2.64 5.58 11.70
2.82 6.51 12.75
35.57
35.38
30.92
36.96
43.49
6.5
60.7
77.9
94.4
Transport of arginine (pmol h 1 m l - i packed cells)
121.0
neither after nitrogen starvation nor after glucose pretreatment. The uptake rates of the amino acids of group III, except for glutamine, and threonine showed a large increase (e.g. methionine 80-fold), but the absolute rates were quite small compared with those of group I. As with glucose-treated cells, it is not clear, therefore, whether specific transport systems for methionine and histidine are "induced" by nitrogen starvation, or whether these amino acids are taken up to some extent by the very active proline and arginine system. The question arises as to whether a common factor is responsible for the increased activity of the amino-acid transport systems under these two different conditions. Nitrogen-starved cells would be expected to possess only small pools of free amino acids. High amounts of soluble carbohydrates would be expected for glucose-fed cells, since almost 50% of the glucose taken up is converted to sucrose (Tanner et al. 1966). In the case of induction by the non-metabolizable analogue 6-deoxy-glucose (Cho et al. 1981), this sugar analogue at least is accumulated to high levels within the cells (Komor et al. 1973). Whether the cellular concentrations of amino acids decrease during glucose or 6-deoxyglucose treatment or the concentrations of soluble carbohydrates increase during nitrogen starvation is not known. To find possible correlations of the increased activity of the aminoacid transport systems with internal concentrations of free sugars and amino acids, changes in concen-
Table 3. Effect of N-starvation on the rate of arginine uptake
and on internal concentration of soluble carbohydrates and amino acids in Chlorella
Exp. I
Cellular concentration (mM)
Time in N-free medium (h) 0
3.5
5
12
Sucrose Arginine Asparagine Glutamic acid + glutamine Total amino acids
2.90 2.35 5.76 3.98
8.66 0.98 1.51 2.60
9.13 0.53 0.86 0.86
-
28.48
20.34
13.46
-
2.5
15.1
21.4
-
1.09 0.21
-
-
13.58 1.18
3.0
-
-
45.5
Transport of arginine (gmol h - i m l - i packed cells) Exp. II Sucrose Glucose Transport of arginine (pmol h - 1 ml 1 packed cells)
tration of these compounds under the various conditions were followed. In Table 2, the cellular concentrations of glucose, sucrose, and amino acids at different times after addition of glucose or glucose plus nitrate are listed. Whereas the arginine transport rate increased 10- to 15-fold within 1 h of glucose treatment, neither a significant increase in sugars nor a corresponding decrease in amino acids has been observed. Also, none of the other amino acids which are not listed in Table 2 changed its concentration during the first hour. Two hours after glucose addition, the concentration of sucrose had doubled and asparagine had decreased to about half. When induction with glucose, however, is carried out in the presence of nitrate, neither the decrease in asparagine nor in total amino acids can be observed, whereas the change in sucrose concentration remains unaffected. But in spite of it, sucrose cannot be regarded as a possible inducer, because its concentration clearly increases subsequent to the induction of the arginine transporter. The same holds for glucose, galactose and mannose (data partly not shown). Under nitrogen starvation (Table 3), on the other hand, the increase in the rate of arginine uptake correlates well with the decrease in concentration of some amino acids, like asparagine, glutamine plus glutamic acid and arginine. This is also true for the increase in sucrose content. Amino acids and sucrose change in concentration before
N. Sauer et al. : Amino-acid transport in algae ,F:
'-2 u
"7 E
% -,D 10
'2O
IE
407 Table 4. Transport of arginine in Chlorella. Comparison of Km and Vmax values. Uptake experiments were performed as described under Material and methods. Cell densities varied from 10 gl packed cells m1-1 at 1 mM L-arginine (specific activity: 6.41.109 Bq-mo1-1) to 0.01 gl packed cells/ml at 0.25 pM Larginine (specific activity: 1.282"1013Bq-mol-1). The Vmax values are average values of 7 experiments Ceils N-starved pretreated cells with glucose
o u
"G o~
5
Km(gM) Vmax (gmol ml- 1 packed cells h - 1)
1.6 104 • 31
1.6 73 _+19
u c o u
'~
0
0
I
2
3
6
g '-
5
Time [ h I
Fig. 1. Comparison of changes in cellular concentrations of asparagine ( o - - o ) , and sucrose ( n - - n ) with the increasing rate for arginine transport (e e). At zero time the Chlorella cells were transferred to N-free medium in the light plus CO 2 ; p.c. = packed cells
a significant induction of arginine uptake can be observed. In the first h o u r after the removal of the N-source, the concentration of asparagine drops to 50% o f the previous value while sucrose doubles (Fig. 1). Thus, it could be concluded that under nitrogen starvation the internal pools of amino acids or sucrose, or some kind of a C / N ratio might control the induction o f the two aminoacid transport systems. Since induction by glucose does not result in the same correlative pattern, the question arose, whether the increased rate o f amino-acid uptake after N-starvation and glucose pretreatment is due to identical systems.
Comparison of arginine transport induced in two different ways. First, the specificity of the differently induced arginine transporters was examined. As already mentioned above (Table 1), with the exception of small differences, the same amino acids are transported at a high rate after both m e t h o d s of induction. Second, in view o f the fact that an i m p o r t a n t aspect concerning the identity of the two transport proteins would be identical K m values, the apparent K m values and the Vm,~ values for arginine uptake o f glucose-treated and N-starved cells are compared (Table 4). The K m values o f 1.6 g M are identical under both conditions, the Vm, ~ values are generally 20-30% lower in N-starved cells. A lowered rate of protein synthesis in these cells might be responsible for this difference.
Table 5. Effect of glucose treatment on transport rates (pmol ml-1 packed cells h-1) of nitrogen-starved cells of Chlorella. Where indicated 10 mM NO~- was present; other conditions see Material and methods Transported compound
N-starved cells (control)
Cells treated with glucose
Cells treated with glucose+ NO3-
Glucose Arginine Proline
0.7 57.0 59.5
6.5 45.6 47.6
47.6 62.7 41.7
Finally, it was investigated whether the two transporters can be induced additively. In Nstarved cells the hexose uptake system of Chlorella is not active. Addition of glucose to such cells also does n o t increase glucose uptake significantly. This indicates that shortage o f internal amino acids limits the synthesis of the hexose transport protein (Fenzl et al. /977). U n d e r these conditions glucose also would not be expected to have any inducing effect on the amino-acid uptake systems. This is indeed the case (Table 5). W h e n these cells are treated, however, with glucose plus nitrate, the rate of glucose uptake increases almost 70-fold (alt h o u g h the rate obtained is only 25-30% of that observed in normal cells after induction). This shows that under these conditions the cells are capable of de-novo protein synthesis started by an inducer such as glucose. U n d e r the same conditions, however, there is no increase in the activity of the arginine transport system (Table 5). Thus, the two treatments, glucose induction and nitrogen starvation are not additive, which indeed supports the conclusion that one and the same transport system is being turned o n by the two treatments.
Comparison of the proline transport system induced in two different ways. The proline uptake system was characterized in the same way as arginine uptake. In Table 6, the apparent K m and the Vm~x
408
N. Sauer et al. : Amino-acid transport in algae
Table 6. Transport of proline in Chlorella. Comparison of K m and Vm~X values. The data were obtained as described in Table 4. The Vm~x values are average values of five experiments
u
"T =o
100
Cells N-starved pretreated cells with glucose K m (p.M) V,.ax (pmol ml 1 packed cells h - 1)
39 94_+22
50
35 47 _+12
I
values for glucose-pretreated and N-starved cells are compared. There was no significant difference between cells treated in either way. The K m value of around 40 gM is, however, more than a factor of 20 higher than that for arginine. As with the arginine system, the Vmax of proline uptake by glucose-induced cells is generally somewhat higher than that of N-starved cells. The fact that the proline system cannot be additively induced (Table 5), and that it shows the same specificity after both methods of induction (Table 1), leads to the same conclusion as with the arginine system: in both cases it is likely that the same transport protein becomes induced.
How does the maximal inside concentration during proline and arginine uptake arise? Uptake of arginine proceeds beyond the concentration equilibrium (Cho et al. 1981). This is demonstrated in greater detail in Table 7. Accumulation ratios of 600- and up to 1200-fold are observed at an initial outside concentration of I m M (see also Cho et al. 1981). The accumulation ratio decreases with increasing outside concentration, a phenomenon generally observed in transport physiology (Komor etal. 1973; Kotyk 1973; Eddy 1982). The maximal accumulation ratios observed are far beyond the electrochemical equilibrium potential of about 130 mV inside negative (Komor and Tanner 1976) and thus not in agreement with a mechanism of arginine moving as a cation in a uniport manner; they rather indicate an Arg +/H + symport.
[
I
I
I
I
60
120 Time [ min]
Flux Rates
[ y m o l h -ImL-Ip.c.]
Net ]nftux
Gross Influx
rain
180
180
rain
57
61
